Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization. The cellular machinery that generates this ability is known collectively as the biological clock. The importance of a detailed understanding of the circadian clock to our understanding of physical and mental health and to the treatment of mental illness rests on the ubiquity of its influence on human mental and physiological processes. These range from circadian changes in basic physiological functions to the clear involvement of circadian rhythms in human work rest cycles and sleep. Human psychiatric illnesses known to be a direct result of clock malfunction include common forms of manic-depressive illness and insomnia. Extensive research in the past has demonstrated the extent and significance of clock control of gene expression and enzyme activities, but in general little is known regarding how clocks control the metabolism of the cells in which they operate. One salient aspect of this regulation is clock control of mRNA abundance via circadian regulation of DNA transcription. We have used subtractive hybridization in the context of circadian control of gene expression to identity genes that are unequivocally under circadian clock control. Recent studies have proven that these genes are regulated at the level of transcription, thereby establishing that there must exist cis-acting regulatory sequences within or adjacent to these genes that are recognized by clock-control factors. In the proposed studies we will use genetic and biochemical screens to identify and characterize these morning specific clock-control factors. Initial studies identified only genes expressed specifically in the morning. We have begun and will complete the identification of genes expressed specifically at other times of day in order to evaluate the extent of clock control of gene expression throughout the day. Using molecular tools that we have developed, we will carry out experiments designed to determine whether, and if so how, these clock-regulated genes and their products are involved in the mechanism or regulation of the biological clock. Specifically, recent experiments from our lab, and also from Aplysia, suggest that there exist certain clock-critical RNAs made in the early morning, and that these encode, soon after their synthesis, clock-critical proteins. We will continue our analysis of morning specific genes in order to identity the clock-critical gene(s). Overall, these studies are a first step toward mapping out the temporal topology of circadian regulation of gene expression, and toward developing a system where clock control can be studied on a broad scale at the molecular level.